Motor proteins and methods for their use

ABSTRACT

The present invention provides high throughput screening system&#39;s for identifying compounds useful in the treatment of cellular proliferation disorders. The method can be performed in plurality simultaneously with fluorescence or absorbance readouts.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/723,595, filed on Nov. 28, 2000, now U.S. Pat. No. 6,548,267 issuedApr. 15, 2003, which is a continuation of U.S. application Ser. No.09/596,541, filed Jun. 16, 2000, now U.S. Pat. No. 6,361,993 issued Mar.26, 2002, which is a continuation in part of U.S. application Ser. No.09/295,612 filed Apr. 20, 1999, now abandoned, which are incorporatedherein by reference.

FIELD OF TIE INVENTION

The invention relates to methods for the identification of compoundsthat modulate the activity of target proteins having motor domains anduse of such methods for the identification of therapeutic agents.

BACKGROUND OF THE INVENTION

The kinesin superfamily is an extended family of related microtubulemotor proteins. It can be classified into at least 8 subfamilies basedon primary amino acid sequence, domain structure, velocity of movement,and cellular function. This family is exemplified by “true” kinesin,which was first isolated from the axoplasm of squid, where it isbelieved to play a role in anterograde axonal transport of vesicles andorganelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)).

Mitotic kinesins are enzymes essential for assembly and fiction of themitotic spindle, but are not generally part of other microtubulestructures. Mitotic kinesins play essential roles during all phases ofmitosis. These enzymes are “molecular motors” that translate energyreleased by hydrolysis of ATP into mechanical force which drives thedirectional movement of cellular cargoes along microtubules. Thecatalytic domain sufficient for this task is a compact structure ofapproximately 340 amino acids. During mitosis, kinesins organizemicrotubules into the bipolar spindle that is the mitotic spindle.Kinesins mediate movement of chromosomes along spindle microtubules, aswell as structural changes in the mitotic spindle associated withspecific phases of mitosis. Experimental perturbation of mitotic kinesinfunction causes malformation or dysfunction of the mitotic spindle,frequently resulting in cell cycle arrest.

Within this functional group of kinesins resides a group of kinesinsfrom several organisms that share significant sequence homology, theKAR3 family of minus end-directed motor proteins. These include, but arenot Limited to, HSET (the human homologue of the KAR3 family);Drosophila melanogaster nonclaret disjunctional (“Dmncd”); C. elegansKlp-3; MmKifCl; XlXCTKS, AtKatA, AtKatB, AtKatC, AnKLPA, SpoKLP2,DdKRPK2, SpoKLP1, ScKAR3, CgCHO2, and the like.

One of the best studied members of this family is ncd. Ncd mutationleads to spindles with splayed poles that are frequently split intomultiple distinct foci, and spurs of microtubules have been observed toproject from the main body of these spindles. This motor and itshomologues are believed to contribute to both the overall structuralintegrity of the spindle and the efficiency of spindle formation byfocusing microtubule minus ends.

HSET has been shown to localize between microtubules in the metaphasespindle of human cells. It has also been shown that HSET is essential toestablish cohesive poles in mouse meiotic spindles. HSET is believed toact antagonistically to the plus end-directed activity of KSP, both invitro and in vivo. These two motor proteins, through cross-linking andoppositely oriented motor activity, are thought to generate awell-ordered framework of microtubule bundles within the spindle. Thiscross-linking activity is important for the overall structural stabilityof the spindle lattice. Thus, the kinesin HSET plays an important rolein the mitotic process. See, e.g., Mountain et al. (1999) J. Cell Biol.147:351; Sawin and Endow (1993) Bioessays, 15:399; Khan et al. (1997) J.Mol. Biol. 270:627; Nakagawa et al. (1997) Proc. Natl. Acad. Sci. USA94:9654; and Hirokawa et al. (1998) Science 279:519.

Defects in function of HSET could be expected to result in cell cyclearrest in mitosis. As such, compounds that modulate the activity of thiskinesin may affect cellular proliferation. The present inventionprovides a novel method to identify such compounds.

SUMMARY OF THE INVENTION

The present invention provides methods to identify candidate agents thatbind to a target protein or act as a modulator of the bindingcharacteristics or biological activity of a target protein. In oneembodiment, the method is performed in plurality simultaneously. Forexample, the method can be performed at the same time on multiple assaymixtures in a multi-well screening plate. Furthermore, in a preferredembodiment, fluorescence or absorbance readouts are utilized todetermine activity. Thus, in one aspect, the invention provides a highthroughput screening system for detecting modulators of activity atarget protein.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate, under conditions that normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to areaction that uses said ADP or phosphate as a substrate under conditionsthat normally allow the ADP or phosphate to be utilized and determiningthe level of activity of the reaction as a measure of the concentrationof ADP or phosphate. A change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

The phrase “use ADP or phosphate” means that the ADP or phosphate aredirectly acted upon by detection reagents. In one case, the ADP, forexample, can be hydrolyzed or can be phosphorylated. As another example,the phosphate can be added to another compound. As used herein, in eachof these cases, ADP or phosphate is acting as a substrate.

Preferably, the target protein either directly or indirectly producesADP or phosphate and comprises a motor domain. More preferably, thetarget protein comprises HSET or a fragment thereof. Most preferably,the target protein comprises SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

Also provided are modulators of the target protein including agents forthe treatment of cellular proliferation, including cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation. Theagents and compositions provided herein can be used in variety ofapplications which include the formulation of sprays, powders, and othercompositions. Also provided herein are methods of treating cellularproliferation disorders such as cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation, for treatingdisorders associated with HSET activity, and for inhibiting HSET.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:1) wherein the startand stop codons are framed.

FIG. 2 shows an embodiment of a particularly preferred target protein(SEQ ID NO:2). The construct contains residues 151 through 510 of thefull length HSET enzyme.

FIG. 3 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:3) wherein the startand stop codons are framed.

FIG. 4 shows an embodiment of another particularly preferred targetprotein (SEQ D NO:4). The construct contains residues 151 through 519 ofthe full length HSET enzyme.

FIG. 5 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:5) wherein the startand stop codons are framed.

FIG. 6 shows an embodiment of another particularly preferred targetprotein (SEQ ID NO:6). The construct contains residues 152 through 519of the full length HSET enzyme.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“ADP” refers to adenosine diphosphate and also includes ADP analogs,including, but not limited to, deoxyadenosine diphosphate (dADP) andadenosine analogs.

“Biologically active” target protein refers to a target protein that hasone or more of kinesin protein's biological activities, including, butnot limited to microtubule stimulated ATPase activity, as tested, e.g.,in an ATPase assay. Biological activity can also be demonstrated in amicrotubule gliding assay or a microtubule binding assay. “ATPaseactivity” refers to ability to hydrolyze ATP. Other activities includepolymerization/depolymerization (effects on microtubule dynamics),binding to other proteins of the spindle, binding to proteins involvedin cell-cycle control, or serving as a substrate to other enzymes, suchas kinases or proteases and specific kinesin cellular activities, suchas chromosome congregation, axonal transport, etc.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a target protein or a fragment thereof or nucleicacid encoding a target protein or a fragment thereof. Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. A biological sample comprises at least one cell,preferably plant or vertebrate. Embodiments include cells obtained froma eukaryotic organism, preferably eukaryotes such as fungi, plants,insects, protozoa, birds, fish, reptiles, and preferably a mammal suchas rat, mice, cow, dog, guinea pig, or rabbit, and most preferably aprimate such as chimpanzees or humans.

A “comparison window” includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appi. Math 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a dendrogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). As ageneral rule, PileUp can align up to 500 sequences, with any singlesequence in the final alignment restricted to a maximum length of 7,000characters.

The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster can then be aligned to the -next most relatedsequence or cluster of aligned sequences. Two clusters of sequences canbe aligned by a simple extension of the pairwise alignment of twoindividual sequences. A series of such pairwise alignments that includesincreasingly dissimilar sequences and clusters of sequences at eachiteration produces the final alignment.

“Variant” applies to both amino acid and nucleic acid sequences. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

Also included within the definition of target proteins of the presentinvention are amino acid sequence variants of wild-type target proteins.These variants fall into one or more of three classes: substitutional,insertional or deletional variants. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe target protein, using cassette or PCR mutagenesis or othertechniques well known in the art, to produce,DNA encoding the variant,and thereafter expressing the DNA in recombinant cell culture. Varianttarget protein fragments having up to about 100-150 amino acid residuesmay be prepared by in vitro synthesis using established techniques.Amino acid sequence variants are characterized by the predeterminednature of the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variation of the target protein aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally occurring analogue, althoughvariants can also be selected which have modified characteristics.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to about 20 amino acids,although considerably longer insertions may be tolerated. Deletionsrange from about 1 to about 20 residues, although in some cases,deletions may be much longer.

Substitutions, deletions, and insertions or any combinations thereof maybe used to arrive at a final derivative. Generally, these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger characteristics may be tolerated in certaincircumstances.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutaric acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) isoleucine (I), Leucine (L), Methionine (M), Vatine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

“Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. Includes cytoskeletal polymers (e.g., actin filaments,microtubules, intermediate filaments, myosin fragments), molecularmotors (e.g., kinesins, myosins, dyneins), cytoskeleton associatedregulatory proteins (e.g., tropomysin, alpha-actinin) and cytoskeletalassociated binding proteins (e.g., microtubules associated proteins,actin binding proteins).

“Cytoskeletal function” refers to biological roles of the cytoskeleton,including but not limited to the providing of structural organization(e.g., microvilli, mitotic spindle) and the mediation of motile eventswithin the cell (e.g., muscle contraction, mitotic chromosome movements,contractile ring formation and function, pseudopodal movement, activecell surface deformations, vesicle formation and translocation.)

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

“High stringency conditions” may be identified by those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (v/v) formamide with 0. 1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5 ×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2 ×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1 ×SSC containing EDTA at 55°C.

“High throughput screening” as used herein refers to an assay whichprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, or plant cells. Both primary cells and cultured cell Lines areincluded in this definition.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. Lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is 50or 100 amino acids in length. This definition also refers to thecomplement of a test sequence, provided that the test sequence has adesignated or substantial identity to a reference sequence. Preferably,the percent identity exists over a region of the sequence that is atleast about 25 nucleotides in length, more preferably over a region thatis 50 or 100 nucleotides in length.

When percentage of sequence identity is used in reference to proteins orpeptides; it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g,. charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. The scoring of conservative substitutions can becalculated according to, e.g., the algorithm of Meyers & Millers,Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, Calif.).

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated target gene, the nucleic acid of interest is separated fromopen reading frames which flank the target gene and encode proteinsother than the target protein. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include fluorescent proteins such as green, yellow, red or bluefluorescent proteins, radioisotopes such as ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g., the polypeptide of SEQ IDNO:2 can be made detectable, e.g., by incorporating a radio-label intothe peptide, and used to detect antibodies specifically reactive withthe peptide).

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5 ×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5 ×Denhardt's solution, 10%dextran sulfate, and 20 ; μg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1 ×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

“Modulators,” “inhibitors,” and “activators of a target protein” referto modulatory molecules identified using in vitro and in vivo assays fortarget protein activity. Such assays include ATPase activity,microtubule gliding, microtubute depolymerizing activity, and bindingactivity such as microtubule binding activity or binding of nucleotideanalogs. Samples or assays that are treated with a candidate agent at atest and control concentration. The control concentration can be zero.If there is a change in target protein activity between the twoconcentrations, this change indicates the identification of a modulator.A change in activity, which can be an increase or decrease, ispreferably a change of at least 20% to 50%, more preferably by at least50% to 75%, more preferably at least 75% to 100%, and more preferably150% to 200%, and most preferably is a change of at least 2 to 10 foldcompared to a control. Additionally, a change can be indicated by achange in binding specificity or substrate.

“Molecular motor” or “motor protein” refers to a molecule that utilizeschemical energy to generate mechanical force. According to oneembodiment, the molecular motor drives the motile properties of thecytoskeleton.

The phrase “motor domain” refers to the domain of a target protein thatconfers membership in the kinesin superfamily of motor proteins througha sequence identity of approximately 35-45% identity to the motor domainof true kinesin.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. For example, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608(1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid probe or oligonucleotide” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, or T) ormodified bases. In addition, the bases in a probe may be joined by alinage other than a phosphodiester bond, so long as it does notinterfere with hybridizatioa. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidine complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. A target protein comprises a polypeptide demonstrated to haveat least microtubule stimulated ATPase activity. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by Nomenclature Commission. Nucleotides, likewise, may be referred toby their commonly accepted single-letter codes, i.e., the one-lettersymbols recommended by the IUPAC-IUB.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA box element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription. A “constitutive” promoter isa promoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is deterninativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised tothe target protein with the amino acid sequence encoded in SEQ ID NO:2can be selected to obtain only those antibodies that are specificallyimmunoreactive with the target protein and not with other proteins,except for polymorphic variants, orthologs, alleles, and closely relatedhomologues of HSET. This selection may be achieved by subtracting outantibodies that cross react with molecules, for example, such as C.elegans unc-104 and human KiflA. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific imnmunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the cytoskeletal system in vivo which can haveapplication in both human and animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding, but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalsound healing, inflammatory and immune disorders such as rheumatoidarthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic disesase such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma, andOsler Webber syndrome.

II. The Target Protein

According to the present invention, a target protein is a molecule thateither directly or indirectly produces ADP or phosphate and thatcomprises a motor domain. In a preferred embodiment, the target proteinis an enzyme having activity which produces ADP and/or phosphate as areaction product. Also included within the definition of the targetproteins are amino acid sequence variants of wild-type target proteins.

Target proteins of-the present invention may also be modified in a wayto form chimeric molecules comprising a fusion of a target protein witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affinity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al., (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Hopp etal. (1988) BioTechnology 6:1204); the KT3 epitope peptide (see, Martineet al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner(1991) 3. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag(see, Lutz-Freyernuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.Target proteins of the present invention are meant to include both theuntagged target protein as well as the chimeric protein wherein thetarget protein has been fused to one or more tag epitopes.

In a particularly preferred embodiment, the target protein comprisesHSET or a fragment thereof.

In another aspect of this invention, the target protein comprises anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, preferably greater than 80%,more preferably greater than 90%, more preferably greater than 95% or,in another embodiment, has 98 to 100% sequence identity with SEQ IDNO:2, SEQ ID NO:4, or SEQ ID NO:6.

In a particularly preferred embodiment, a fragment of the HSET proteincomprising a portion of its hydrolytically active “motor” domain isused. This motor domain has been cloned and expressed in bacteria suchthat large quantities of biochemicaLly active, substantially pureprotein are available. Preferably, the target protein comprises an aminoacid sequence which has greater than 70% sequence identity with SEQ IDNO:2, SEQ ID NO:4, or SEQ ID NO:6, preferably greater than 800%, morepreferably greater than 90%, more preferably greater than 95% or, inanother embodiment, has 98 to 100% sequence identity with SEQ D NO:2,SEQ ID NO:4, or SEQ ID NO:6.

A particularly preferred embodiment is drawn to a fragment of the HSETprotein SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. More preferably, thisfragment is tagged at the C-terminus with a myc epitope and 6histidines. More preferably, this fragment is tagged at the N-terminuswith a T7 epitope and at the C-terminus with a myc epitope and 6histidines.

In one aspect, the nucleic acids provided herein are defined by theproteins encoded thereby. A preferred embodiment of the invention isdrawn to an isolated nucleic acid sequence encoding a microtubule motorprotein, wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 70% sequenceidentity with SEQ ID NO:2, SEQ ED NO:4 or SEQ ID NO:6, preferablygreater than 80%, more preferably greater than 90%, more preferablygreater than 95% or, in another embodiment, has 98 to 100% sequenceidentity with SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In oneembodiment, the nucleic acid encodes HSET or a fragment thereof. Inanother embodiment, the nucleic acid encodes SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6.

In one embodiment, the nucleic acid comprises a sequence which has oneor more of the following characteristics: greater than 55 or 60%sequence identity with SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO: 5,preferably greater than 70%, more preferably greater than 80%, morepreferably greater than 90 or 95% or, in another embodiment, has 98 to100% sequence identity with SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. Inanother embodiment provided herein, the nucleic acid hybridizes understringent conditions to a nucleic acid having a sequence orcomplementary sequence of SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO:5. Inanother embodiment, the nucleic acid has a nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5. As described above, when describingthe nucleotide in terms of SEQ ID NO:I, SEQ ID NO:3, or SEQ ID NO:5, thesequence identity may be slightly lower due to the degeneracy in thegenetic code.

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the protein.

Numerous suitable methods for recombinant protein expression, includinggeneration of expression vectors, generation of fusion proteins,introducing expression vectors into host cells, protein expression inhost cells, and purifications methods are known to those in the art.

In a preferred embodiment, the target proteins are purified for use inthe assays to provide substantially pure samples. Alternatively, thetarget protein need not be substantially pure as long as the samplecomprising the target protein is substantially free of other componentsthat can contribute to the production of ADP or phosphate.

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocussing. For example,the target protein can be purified using a standard anti-target antibodycolumn. Ultrafiltration and diafiltration techniques, in conjunctionwith protein concentration, are also useful.

Either naturally occurring or recombinant target protein can be purifiedfor use in functional assays. The target protein may be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra). Apreferred method of purification is use of Ni-NTA agarose (Qiagen).

Suitable purification schemes for some specific kinesins are outlined inU.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby expresslyincorporated herein in its entirety for all purposes.

The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays provided herein such as those which assay ATPaseactivity or microtubule binding activity. Biologically active targetprotein is useful for identifying modulators of target protein orfragments thereof and kinesin superfamily members using in vitro assayssuch as microtubute gliding assays, ATPase assays (Kodama et al., J.Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA90:5209-5213 (1993)), and binding assays including microtubule bindingassays (Vale et al., Cell 42:39-50 (1995)), as described in detailbelow.

III. Assays for Modulators of the Target Protein

A. Functional Assays

Assays that can be used to test for modulators of the target proteininclude a variety of in vitro or in vivo assays, e.g., microtubulegliding assays, binding assays such as microtubule binding assays,microtubule depolymerization assays, and ATPase assays (Kodamna et al.,J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci.USA 90: 5209-5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115(1995); (Vale et al., Cell 42:39-50 (1985)).

Modulation is tested by screening for candidate agents capable ofmodulating the activity of the target protein comprising the steps ofcombining a candidate agent with the target protein, as above, anddetermining an alteration in the biological activity of the targetprotein. Thus, in this embodiment, the candidate agent should both bindto the target protein (although this may not be necessary), and alterits biological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods and in vivo screening of cellsfor alterations in cell cycle distribution, cell viability, or for thepresence, morphology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

In a preferred embodiment, molecular motor activity is measured by themethods disclosed in Ser. No. 09/314,464, filed May 18, 1999, entitled“Compositions and assay utilizing ADP or phosphate for detecting proteinmodulators”, which is incorporated herein by reference in its entirety.More specifically, this assay detects modulators of any aspect of akinesin motor function ranging from interaction with microtubules to thehydrolysis of ATP. ADP or phosphate is used as the readout for proteinactivity.

There are a number of enzymatic assays known in the art which use ADP asa substrate. For example, kinase reactions such as pyruvate kinases areknown. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31 (1970). Thisis a preferred method in that it allows the regeneration of ATP. In oneembodiment, the level of activity of the enzymatic reaction isdetermined directly. In a preferred embodiment, the level of activity ofthe enzymatic reaction which uses ADP as a substrate is measuredindirectly by being coupled to another reaction For example, in oneembodiment, the method further comprises a lactate dehydrogenasereaction under conditions which normally allow the oxidation of NADH,wherein said lactate dehydrogenase reaction is dependent on the pyruvatekinase reaction. Measurement of enzymatic reactions by coupling is knownin the art. Furthermore, there are a number of reactions which utilizephosphate. Examples of such reactions include a purine nucleosidephosphorylase reaction. This reaction can be measured directly orindirectly. A particularly preferred embodiments utilizes the pyruvatekinase/lactate dehydrogenase system.

In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example, by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. One method of quantifying the phosphomolybdateis with malachite green. Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

In addition, target protein activity can be examined by determiningmodulation of target protein in vitro using cultured cells. The cellsare treated with a candidate agent and the effect of such agent on thecells is then determined either directly or by examining relevantsurrogate markers. For example, characteristics such as mitotic spindlemorphology and cell cycle distribution can be used to determine theeffect.

Thus, in a preferred embodiment, the methods comprise combining a targetprotein and a candidate agent, and determining the effect of thecandidate agent on the target protein. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

As will be appreciated by those in the art, the components may be addedin buffers and reagents to assay target protein activity and giveoptimal signals. Since the methods allow kinetic measurements, theincubation periods can be optimized to give adequate detection signalsover the background.

In a preferred embodiment, an antifoam or a surfactant is included inthe assay mixture. Suitable antifoams include, but are not limited to,antifoam 289 (Sigma). Suitable surfactants include, but are not limitedto, Tween, Tritons, including Triton X-100, saponins, andpolyoxyethylene ethers. Generally, the antifoams, detergents, orsurfactants are added at a range from about 0.01 ppm to about 10 ppm.

A preferred assay design is also provided. In one aspect, the inventionprovides a multi-time-point (kinetic) assay, with at least two datapoints being preferred. In the case of multiple measurements, theabsolute rate of the protein activity can be determined.

B. Binding Assays

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget protein, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the candidate agent and the binding moiety, with the bindingmoiety displacing the candidate agent.

Competitive screening assays may be done by combining the target proteinand a drug candidate in a first sample. A second sample comprises acandidate agent, the target protein and a compound that is known tomodulate the target protein. This may be performed in either thepresence or absence of microtubules. The binding of the candidate agentis determined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the target protein and potentially modulating its activity.That is, if the binding of the candidate agent is different in thesecond sample relative to the first sample, the candidate agent iscapable of binding to the target protein.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to the target proteinfor a time sufficient to allow binding. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0. 1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to the target protein and thus is capable ofbinding to, and potentially modulating, the activity of the targetprotein. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the washsolution indicates displacement by the agent. Alternatively, if thecandidate agent is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound tothe target protein with a higher affinity. Thus, if the candidate agentis labeled, the presence of the label on the support, coupled with alack of competitor binding, may indicate the candidate agent is capableof binding to the target protein.

C. Candidate Agents

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. In a preferred embodiment,the candidate agents are organic chemical moieties, a wide variety ofwhich are available in the literature.

D. Other Assay Components

The assays provided utilize target protein as defined herein. In oneembodiment, portions of target protein are utilized; in a preferredembodiment, portions having target protein activity as described hereinare used. In addition, the assays described herein may utilize eitherisolated target proteins or cells or animal models comprising the targetproteins.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Also,reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

IV. Applications

The methods of the invention are used to identify compounds useful inthe treatment of cellular proliferation diseases. Disease states whichcan be treated by the methods and compositions provided herein include,but are not limited to, cancer (further discussed below), autoimmunedisease, arthritis, graft rejection, inflammatory bowel disease,proliferation induced after medical procedures, including, but notlimited to, surgery, angioplasty, and the like. It is appreciated thatin some cases the cells may not be in a hyper or hypo proliferationstate (abnormal state) and still require treatment. For example, duringwound healing, the cells may be proliferating “normally”, butproliferation enhancement may be desired. Similarly, as discussed above,in the agriculture arena, cells may be in a “normal” state, butproliferation modulation may be desired to enhance a crop by directlyenhancing growth of a crop, or by inhibiting the growth of a plant ororganism which adversely affects the crop. Thus, in one embodiment, theinvention herein includes application to cells or individuals afflictedor impending affliction with any one of these disorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not Limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcorna, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, tymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nepbroblastoma], tymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinorna,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myetoma, malignant giant cell tumorchordoma, osteochronfrorna (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meminges (meningioma,meningiosarcoma, gliomnatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gvnecologicaluterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcorna], fallopian tubes (carcinoma); Hematologic:blood (myeloid leukemia [acute and chronic], acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myeLodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma,basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibrorna, keloids, psoriasis;and Adrenal lands: neuroblastoma. Thus, the term “cancerous cell” asprovided herein, includes a cell afflicted by any one of the aboveidentified conditions.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant admnistration of atherapeutically effective dose of the candidate agents of the inventionto a cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those used in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Candidate agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0. 1-100 wt. %. The agents maybe administered alone orin combination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the phrmaceutical compositions are in a watersoluble form, such as pharmaceutically acceptable salts, which is meantto include both acid and base addition salts.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and a oilsand fats. Stabilizing agents, wetting and emulsifing agents, salts forvarying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The administration of the candidate agents of the present invention canbe done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulinonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the candidate agents may bedirectly applied as a solution or spray.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for ATPase assays, microtubule gliding assays, or microtubulebinding assays.

V. Examples

This assay is based on detection of ADP production from a targetprotein's microtubule stimulated ATPase. ATP production is monitored bya coupled enzyme system consisting of pyruvate kinase and lactatedehydrogenase. Under the assay conditions described below, pyruvatekinase catalyzes the conversion of ADP and phosphoenol pyruvate topyruvate and ATP. Lactate dehydrogenase then catalyzes theoxidation-reduction reaction of pyruvate and NADH to lactate and NAD+.Thus, for each molecule of ADP produced, one molecule of NADH isconsumed. The amount of NADH in the assay solution is monitored bymeasuring light absorbance at a wavelength of 340 mm

The final 25 μl assay solution consists of the following: 5 μg/ml targetprotein, 30 μg/ml microtubules, 5 μM Taxol, 0.8 mM NADH, 1.5 mMphosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactatedehydrogenase, 25 mM Pipes/KOH pH 6.8, 2mM MgCl₂, 1 mM EGTA,1 mM MDTT,0.1 mg/ml BSA, 0.001% antifoam 289, and 1 mM ATP.

Potential candidate agents are dissolved in DMSO at a concentration ofabout 1 mg/ml and 0.5 μl of each chemical solution is dispensed into asingle well of a clear 384 well plate. Each of the 384 wells are thenfilled with 20 μl of a solution consisting of all of the assaycomponents described above except for ATP. The plate is agitated at ahigh frequency. To start the assay, 5 μl of a solution containing ATP isadded to each well. The plate is agitated and the absorbance is read at340 nm over various time intervals. The assay is run at roomtemperature.

The assay components and the performance of the assay are optimizedtogether to match the overall read time with the rate of the targetprotein's ADP production. The read time should be long enough for therate of NADH consumption to reach steady state beyond an initial lagtime of several seconds.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

6 1 1085 DNA Human 1 atgaggaact caagggcaac atccgtgtat tctgccgggtccgccctgtc ctgccggggg 60 agcccactcc accccctggc ctcctcctgt ttccctctggccctggtggg ccctctgatc 120 ctccaacccg ccttagcctc tcccggtctg acgagcggcgtgggaccctg agtggggcac 180 cagctccccc aactcgccat gatttttcct ttgaccgggtattcccacca ggaagtggac 240 aggatgaagt gtttgaagag attgccatgc ttgtccagtcagccctggat ggctatccag 300 tatgcatctt tgcctatggc cagacaggca gtggcaagaccttcacaatg gagggtgggc 360 ctgggggaga cccccagttg gaggggctga tccctcgggccctgcggcac ctcttctctg 420 tggctcagga gctgagtggt cagggctgga cctacagctttgtagcaagc tacgtagaga 480 tctacaatga gactgtccgg gacctgctgg ccactggaacccggaagggt caagggggcg 540 agtgtgagat tcgccgtgca gggccaggga gtgaggagctcactgtcacc aatgctcgat 600 atgtccctgt ctcctgtgag aaagaagtgg acgccctgcttcatctggcc cgccagaatc 660 gggctgtggc ccgcacagcc cagaatgaac ggtcatcacgcagccacagt gtattccagc 720 tacagatttc tggggagcac tccagccgag gcctgcagtgtggggccccc ctcagtcttg 780 tggacctggc cgggagtgag cgacttgacc ccggcttagccctcggcccc ggggagcggg 840 aacgccttcg ggaaacacag gccattaaca gcagcctgtccacgctgggg ctggttatca 900 tggccctgag caacaaggag tcccacgtgc cttaccggaacagcaaactg acctacctgc 960 tgcagaactc tctgggtggt agtgctaaga tgctcatgtttgtgaacatt tctccactgg 1020 aagagaacgt ctccgagtcc ctcaactctc tacgctttgcctccaaggtg aaccagtgtg 1080 tttga 1085 2 361 PRT Human 2 Met Gln Glu LeuLys Gly Asn Ile Arg Val Phe Cys Arg Val Arg Pro 1 5 10 15 Val Leu ProGly Glu Pro Thr Pro Pro Pro Gly Leu Leu Leu Phe Pro 20 25 30 Ser Gly ProGly Gly Pro Ser Asp Pro Pro Thr Arg Leu Ser Leu Ser 35 40 45 Arg Ser AspGlu Arg Arg Gly Thr Leu Ser Gly Ala Pro Ala Pro Pro 50 55 60 Thr Arg HisAsp Phe Ser Phe Asp Arg Val Phe Pro Pro Gly Ser Gly 65 70 75 80 Gln AspGlu Val Phe Glu Glu Ile Ala Met Leu Val Gln Ser Ala Leu 85 90 95 Asp GlyTyr Pro Val Cys Ile Phe Ala Tyr Gly Gln Thr Gly Ser Gly 100 105 110 LysThr Phe Thr Met Glu Gly Gly Pro Gly Gly Asp Pro Gln Leu Glu 115 120 125Gly Leu Ile Pro Arg Ala Leu Arg His Leu Phe Ser Val Ala Gln Glu 130 135140 Leu Ser Gly Gln Gly Trp Thr Tyr Ser Phe Val Ala Ser Tyr Val Glu 145150 155 160 Ile Tyr Asn Glu Thr Val Arg Asp Leu Leu Ala Thr Gly Thr ArgLys 165 170 175 Gly Gln Gly Gly Glu Cys Glu Ile Arg Arg Ala Gly Pro GlySer Glu 180 185 190 Glu Leu Thr Val Thr Asn Ala Arg Tyr Val Pro Val SerCys Glu Lys 195 200 205 Glu Val Asp Ala Leu Leu His Leu Ala Arg Gln AsnArg Ala Val Ala 210 215 220 Arg Thr Ala Gln Asn Glu Arg Ser Ser Arg SerHis Ser Val Phe Gln 225 230 235 240 Leu Gln Ile Ser Gly Glu His Ser SerArg Gly Leu Gln Cys Gly Ala 245 250 255 Pro Leu Ser Leu Val Asp Leu AlaGly Ser Glu Arg Leu Asp Pro Gly 260 265 270 Leu Ala Leu Gly Pro Gly GluArg Glu Arg Leu Arg Glu Thr Gln Ala 275 280 285 Ile Asn Ser Ser Leu SerThr Leu Gly Leu Val Ile Met Ala Leu Ser 290 295 300 Asn Lys Glu Ser HisVal Pro Tyr Arg Asn Ser Lys Leu Thr Tyr Leu 305 310 315 320 Leu Gln AsnSer Leu Gly Gly Ser Ala Lys Met Leu Met Phe Val Asn 325 330 335 Ile SerPro Leu Glu Glu Asn Val Ser Glu Ser Leu Asn Ser Leu Arg 340 345 350 PheAla Ser Lys Val Asn Gln Cys Val 355 360 3 1113 DNA Human 3 atgcaggaactcaagggcaa catccgtgta ttctgccggg tccgccctgt cctgccgggg 60 gagcccactccaccccctgg cctcctcctg tttccctctg gccctggtgg gccctctgat 120 cctccaacccgccttagcct ctcccggtct gacgagcggc gtgggaccct gagtggggca 180 ccagctcccccaactcgcca tgatttttcc tttgaccggg tattcccacc aggaagtgga 240 caggatgaagtgtttgaaga gattgccatg cttgtccagt cagccctgga tggctatcca 300 gtatgcatctttgcctatgg ccagacaggc agtggcaaga ccttcacaat ggagggtggg 360 cctgggggagacccccagtt ggaggggctg atccctcggg ccctgcggca cctcttctct 420 gtggctcaggagctgagtgg tcagggctgg acctacagct ttgtagcaag ctacgtagag 480 atctacaatgagactgtccg ggacctgctg gccactggaa cccggaaggg tcaagggggc 540 gagtgtgagattcgccgtgc agggccaggg agtgaggagc tcactgtcac caatgctcga 600 tatgtccctgtctcctgtga gaaagaagtg gacgccctgc ttcatctggc ccgccagaat 660 cgggctgtggcccgcacagc ccagaatgaa cggtcatcac gcagccacag tgtattccag 720 ctacagatttctggggagca ctccagccga ggcctgcagt gtggggcccc cctcagtctt 780 gtggacctggccgggagtga gcgacttgac cccggcttag ccctcggccc cggggagcgg 840 gaacgccttcgggaaacaca ggccattaac agcagcctgt ccacgctggg gctggttatc 900 atggccctgagcaacaagga gtcccacgtg ccttaccgga acagcaaact gacctacctg 960 ctgcagaactctctgggtgg tagtgctaag atgctcatgt ttgtgaacat ttctccactg 1020 gaagagaacgtctccgagtc cctcaactct ctacgctttg cctccaaggt gaaccagtgt 1080 gttattggtactgctcaggc caacaggaag tga 1113 4 370 PRT Human 4 Met Gln Glu Leu Lys GlyAsn Ile Arg Val Phe Cys Arg Val Arg Pro 1 5 10 15 Val Leu Pro Gly GluPro Thr Pro Pro Pro Gly Leu Leu Leu Phe Pro 20 25 30 Ser Gly Pro Gly GlyPro Ser Asp Pro Pro Thr Arg Leu Ser Leu Ser 35 40 45 Arg Ser Asp Glu ArgArg Gly Thr Leu Ser Gly Ala Pro Ala Pro Pro 50 55 60 Thr Arg His Asp PheSer Phe Asp Arg Val Phe Pro Pro Gly Ser Gly 65 70 75 80 Gln Asp Glu ValPhe Glu Glu Ile Ala Met Leu Val Gln Ser Ala Leu 85 90 95 Asp Gly Tyr ProVal Cys Ile Phe Ala Tyr Gly Gln Thr Gly Ser Gly 100 105 110 Lys Thr PheThr Met Glu Gly Gly Pro Gly Gly Asp Pro Gln Leu Glu 115 120 125 Gly LeuIle Pro Arg Ala Leu Arg His Leu Phe Ser Val Ala Gln Glu 130 135 140 LeuSer Gly Gln Gly Trp Thr Tyr Ser Phe Val Ala Ser Tyr Val Glu 145 150 155160 Ile Tyr Asn Glu Thr Val Arg Asp Leu Leu Ala Thr Gly Thr Arg Lys 165170 175 Gly Gln Gly Gly Glu Cys Glu Ile Arg Arg Ala Gly Pro Gly Ser Glu180 185 190 Glu Leu Thr Val Thr Asn Ala Arg Tyr Val Pro Val Ser Cys GluLys 195 200 205 Glu Val Asp Ala Leu Leu His Leu Ala Arg Gln Asn Arg AlaVal Ala 210 215 220 Arg Thr Ala Gln Asn Glu Arg Ser Ser Arg Ser His SerVal Phe Gln 225 230 235 240 Leu Gln Ile Ser Gly Glu His Ser Ser Arg GlyLeu Gln Cys Gly Ala 245 250 255 Pro Leu Ser Leu Val Asp Leu Ala Gly SerGlu Arg Leu Asp Pro Gly 260 265 270 Leu Ala Leu Gly Pro Gly Glu Arg GluArg Leu Arg Glu Thr Gln Ala 275 280 285 Ile Asn Ser Ser Leu Ser Thr LeuGly Leu Val Ile Met Ala Leu Ser 290 295 300 Asn Lys Glu Ser His Val ProTyr Arg Asn Ser Lys Leu Thr Tyr Leu 305 310 315 320 Leu Gln Asn Ser LeuGly Gly Ser Ala Lys Met Leu Met Phe Val Asn 325 330 335 Ile Ser Pro LeuGlu Glu Asn Val Ser Glu Ser Leu Asn Ser Leu Arg 340 345 350 Phe Ala SerLys Val Asn Gln Cys Val Ile Gly Thr Ala Gln Ala Asn 355 360 365 Arg Lys370 5 1110 DNA Human 5 atggaactca agggcaacat ccgtgtattc tgccgggtccgccctgtcct gccgggggag 60 cccactccac cccctggcct cctcctgttt ccctctggccctggtgggcc ctctgatcct 120 ccaacccgcc ttagcctctc ccggtctgac gagcggcgtgggaccctgag tggggcacca 180 gctcccccaa ctcgccatga tttttccttt gaccgggtattcccaccagg aagtggacag 240 gatgaagtgt ttgaagagat tgccatgctt gtccagtcagccctggatgg ctatccagta 300 tgcatctttg cctatggcca gacaggcagt ggcaagaccttcacaatgga gggtgggcct 360 gggggagacc cccagttgga ggggctgatc cctcgggccctgcggcacct cttctctgtg 420 gctcaggagc tgagtggtca gggctggacc tacagctttgtagcaagcta cgtagagatc 480 tacaatgaga ctgtccggga cctgctggcc actggaacccggaagggtca agggggcgag 540 tgtgagattc gccgtgcagg gccagggagt gaggagctcactgtcaccaa tgctcgatat 600 gtccctgtct cctgtgagaa agaagtggac gccctgcttcatctggcccg ccagaatcgg 660 gctgtggccc gcacagccca gaatgaacgg tcatcacgcagccacagtgt attccagcta 720 cagatttctg gggagcactc cagccgaggc ctgcagtgtggggcccccct cagtcttgtg 780 gacctggccg ggagtgagcg acttgacccc ggcttagccctcggccccgg ggagcgggaa 840 cgccttcggg aaacacaggc cattaacagc agcctgtccacgctggggct ggttatcatg 900 gccctgagca acaaggagtc ccacgtgcct taccggaacagcaaactgac ctacctgctg 960 cagaactctc tgggtggtag tgctaagatg ctcatgtttgtgaacatttc tccactggaa 1020 gagaacgtct ccgagtccct caactctcta cgctttgcctccaaggtgaa ccagtgtgtt 1080 attggtactg ctcaggccaa caggaagtga 1110 6 369PRT Human 6 Met Glu Leu Lys Gly Asn Ile Arg Val Phe Cys Arg Val Arg ProVal 1 5 10 15 Leu Pro Gly Glu Pro Thr Pro Pro Pro Gly Leu Leu Leu PhePro Ser 20 25 30 Gly Pro Gly Gly Pro Ser Asp Pro Pro Thr Arg Leu Ser LeuSer Arg 35 40 45 Ser Asp Glu Arg Arg Gly Thr Leu Ser Gly Ala Pro Ala ProPro Thr 50 55 60 Arg His Asp Phe Ser Phe Asp Arg Val Phe Pro Pro Gly SerGly Gln 65 70 75 80 Asp Glu Val Phe Glu Glu Ile Ala Met Leu Val Gln SerAla Leu Asp 85 90 95 Gly Tyr Pro Val Cys Ile Phe Ala Tyr Gly Gln Thr GlySer Gly Lys 100 105 110 Thr Phe Thr Met Glu Gly Gly Pro Gly Gly Asp ProGln Leu Glu Gly 115 120 125 Leu Ile Pro Arg Ala Leu Arg His Leu Phe SerVal Ala Gln Glu Leu 130 135 140 Ser Gly Gln Gly Trp Thr Tyr Ser Phe ValAla Ser Tyr Val Glu Ile 145 150 155 160 Tyr Asn Glu Thr Val Arg Asp LeuLeu Ala Thr Gly Thr Arg Lys Gly 165 170 175 Gln Gly Gly Glu Cys Glu IleArg Arg Ala Gly Pro Gly Ser Glu Glu 180 185 190 Leu Thr Val Thr Asn AlaArg Tyr Val Pro Val Ser Cys Glu Lys Glu 195 200 205 Val Asp Ala Leu LeuHis Leu Ala Arg Gln Asn Arg Ala Val Ala Arg 210 215 220 Thr Ala Gln AsnGlu Arg Ser Ser Arg Ser His Ser Val Phe Gln Leu 225 230 235 240 Gln IleSer Gly Glu His Ser Ser Arg Gly Leu Gln Cys Gly Ala Pro 245 250 255 LeuSer Leu Val Asp Leu Ala Gly Ser Glu Arg Leu Asp Pro Gly Leu 260 265 270Ala Leu Gly Pro Gly Glu Arg Glu Arg Leu Arg Glu Thr Gln Ala Ile 275 280285 Asn Ser Ser Leu Ser Thr Leu Gly Leu Val Ile Met Ala Leu Ser Asn 290295 300 Lys Glu Ser His Val Pro Tyr Arg Asn Ser Lys Leu Thr Tyr Leu Leu305 310 315 320 Gln Asn Ser Leu Gly Gly Ser Ala Lys Met Leu Met Phe ValAsn Ile 325 330 335 Ser Pro Leu Glu Glu Asn Val Ser Glu Ser Leu Asn SerLeu Arg Phe 340 345 350 Ala Ser Lys Val Asn Gln Cys Val Ile Gly Thr AlaGln Ala Asn Arg 355 360 365 Lys

What is claimed is:
 1. A kit for screening for modulators of a motorprotein, comprising a protein, wherein said protein comprises the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 and hasmicrotubule stimulated ATPase activity; and instructions for testing foractivity of said protein.
 2. The kit of claim 1, wherein the proteincomprises the amino acid sequence of SEQ ID NO:2.
 3. The kit of claim 1,wherein the protein comprises the amino acid sequence of SEQ ID NO:4. 4.The kit of claim 1, wherein the protein comprises the amino acidsequence of SEQ ID NO:6.
 5. The kit of claim 1, further comprisingreaction tubes.
 6. The kit of claim 1, further comprising a stationarymultiwell plate.
 7. The kit of claim 6, wherein the stationary multiwellplate is a 384-well microtiter plate.
 8. The kit of claim 1, furthercomprising an enzyme system for monitoring ADP or phosphate level. 9.The kit of claim 8, wherein the enzyme system comprises pyruvate kinaseand lactate dehydrogenase.
 10. The kit of claim 8, wherein the enzymesystem comprises a luciferin-luciferase system.